High-throughput biochemical profiling reveals sequence determinants of dCas9 off-target binding and unbinding

The bacterial adaptive immune system CRISPR–Cas9 has been appropriated as a versatile tool for editing genomes, controlling gene expression, and visualizing genetic loci. To analyze Cas9’s ability to bind DNA rapidly and specifically, we generated multiple libraries of potential binding partners for...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2017-05, Vol.114 (21), p.5461-5466
Main Authors: Boyle, Evan A., Andreasson, Johan O. L., Chircus, Lauren M., Sternberg, Samuel H., Wu, Michelle J., Guegler, Chantal K., Doudna, Jennifer A., Greenleaf, William J.
Format: Article
Language:English
Subjects:
Adaptive systems
Bacteria
Bacterial Proteins - metabolism
Binding
Biochemistry
Biological Sciences
Combinatorial analysis
CRISPR
CRISPR-Associated Protein 9
Deoxyribonucleic acid
Determinants
DNA
DNA - metabolism
DNA sequencing
Editing
Endonucleases - metabolism
Flow
Gene expression
Genomes
gRNA
High-Throughput Screening Assays
Immune system
Kinetics
Libraries
Loci
Mathematical models
Nuclease
Nucleotides
Proteins
Ribonucleic acid
RNA
RNA, Guide, CRISPR-Cas Systems - metabolism
Thermodynamic properties
Thermodynamics
Time
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Summary:The bacterial adaptive immune system CRISPR–Cas9 has been appropriated as a versatile tool for editing genomes, controlling gene expression, and visualizing genetic loci. To analyze Cas9’s ability to bind DNA rapidly and specifically, we generated multiple libraries of potential binding partners for measuring the kinetics of nuclease-dead Cas9 (dCas9) interactions. Using a massively parallel method to quantify protein–DNA interactions on a high-throughput sequencing flow cell, we comprehensively assess the effects of combinatorial mismatches between guide RNA (gRNA) and target nucleotides, both in the seed and in more distal nucleotides, plus disruption of the protospacer adjacent motif (PAM). We report two consequences of PAM-distal mismatches: reversal of dCas9 binding at long time scales, and synergistic changes in association kinetics when other gRNA–target mismatches are present. Together, these observations support a model for Cas9 specificity wherein gRNA–DNA mismatches at PAM-distal bases modulate different biophysical parameters that determine association and dissociation rates. The methods we present decouple aspects of kinetic and thermodynamic properties of the Cas9–DNA interaction and broaden the toolkit for investigating off-target binding behavior.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.1700557114